Heat is a powerful stressor for fish living in natural and artificial environments. Understanding the effects of heat stress on the physiological processes of fish is essential for better aquaculture and fisheries management. In this experiment, a heating rod was used to increase the temperature at 2°C/h to study the changes of energy allocation (CEA) and energy metabolity-related enzyme activities, including pepsin, trypsin, amylase, lipase, acid phosphatase, lactate dehydrogenase, alanine aminotransferase, glutamic oxalic aminotransferase and energy reserve (Ea), energy expenditure (ETS), in juvenile yellowfin tuna cells under acute temperature stress. The results showed that the Ea of juvenile yellowfin tuna muscles in response to high temperature (34°C) was significantly lower than that of the control (28°C), and it also increased ETS. At 6 h, CEA decreased slightly in the high-temperature group, but, the difference in CEA between 24 h and 0 h decreased. After heat stress for 6 h, the activities of acid phosphatase (ACP), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and glutamic oxalacetic transaminase (AST) increased, indicating that the metabolic rate was accelerated. After heat stress for 24 h, the activity of ALT decreased, indicating that with time elapsed, the activities of some protein metabolizing enzymes increased, and some decreased. In this study, digestive enzymes, trypsin and lipase increased gradually. After heat stress, Ea and Ec change significantly. Yellowfin tuna muscles use lipids in response to sharp temperature increases at high temperatures, red muscles respond to temperature changes by increasing energy in the early stages, but not nearly as much, and white muscles reduce lipids.